The isolation and characterization of human mammary stem cells and the elucidation of the mechanisms that govern their self-renewal and differentiation could offer important insights into normal mammary development and carcinogenesis. Although the existence of mammary stem cells has been suggested by cellular marking and transplantation studies in mice, their identification and isolation have been hindered by the lack of specific markers for these cells and suitable in vitro systems for their cultivation. We have developed an in vitro culture system in which human mammary epithelial cells (HMEC) can be cultured in suspension as "mammospheres". We demonstrated that these mammospheres are highly enriched in mammary stem/progenitor cells. These cells form human ductal alveolar structures when implanted into the cleared fat pads of NOD/SCID mice. Gene expression profiles of mammosphere derived cells show significant overlap with stem cells derived from other tissue. We propose to prospectively isolate human mammary stem cells by immunosorting, utilizing markers expressed on mammosphere-derived cells, which are also expressed on other stem cells. The stem cell properties of self-renewal and multi-lineage differentiation will be assayed utilizing both in vitro and in vivo assays. We propose to utilize this new culture system to elucidate the pathways involved in cell fate determination of human mammary stem/progenitor cells. We postulate the involvement of self-renewal pathways common to other stem cells, including Notch, Wnt, LIF, and Bmi-1. We propose to examine the effects of stimulation or inhibition of these pathways on self-renewal and lineage specific differentiation, utilizing both in vitro assays and the NOD/SCID mouse model. We will also use these systems to study interactions between stem/progenitor cells and mammary stromal cells. These studies should help elucidate pathways that regulate self-renewal and lineage specific commitment of human mammary stem/progenitor cells. This should provide important insights into the processes that regulate normal mammary development, processes that may be altered during mammary carcinogenesis.